U.S. patent number 4,546,500 [Application Number 06/352,585] was granted by the patent office on 1985-10-15 for fabrication of living blood vessels and glandular tissues.
This patent grant is currently assigned to Massachusetts Institute of Technology. Invention is credited to Eugene Bell.
United States Patent |
4,546,500 |
Bell |
* October 15, 1985 |
Fabrication of living blood vessels and glandular tissues
Abstract
A method and apparatus for producing a vessel-equivalent
prosthesis is described. A contractile agent such as fibroblast
cells, smooth muscle cells or platelets is incorporated into a
collagen lattice and contracts the lattice axially around an inner
core. After the structure has set, additional layers may be formed
in an ordered manner depending on the intended function of the
prosthesis. Alternatively, all the layers may be formed
concurrently. A plastic mesh sleeve is sandwiched between layers or
embedded within the smooth muscle cell layer to reinforce the
structure and provide sufficient elasticity to withstand
intravascular pressure.
Inventors: |
Bell; Eugene (Dedham, MA) |
Assignee: |
Massachusetts Institute of
Technology (Cambridge, MA)
|
[*] Notice: |
The portion of the term of this patent
subsequent to September 10, 2002 has been disclaimed. |
Family
ID: |
26948924 |
Appl.
No.: |
06/352,585 |
Filed: |
February 26, 1982 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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261928 |
May 8, 1981 |
4539716 |
|
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Current U.S.
Class: |
435/1.1; 623/901;
623/921 |
Current CPC
Class: |
A61F
2/022 (20130101); A61L 27/24 (20130101); A61F
2/06 (20130101); A61L 27/507 (20130101); A61F
2/062 (20130101); A61L 27/36 (20130101); A61F
2/06 (20130101); Y10S 623/901 (20130101); Y10S
623/921 (20130101); A61F 2310/00365 (20130101) |
Current International
Class: |
A61F
2/06 (20060101); A61F 2/02 (20060101); A61L
27/50 (20060101); A61L 27/24 (20060101); A61L
27/00 (20060101); A61F 2/00 (20060101); A61F
001/00 () |
Field of
Search: |
;3/1,1.4 ;435/240-241,1
;128/DIG.8 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Sarber, R. et al., "Regulation of Proliferation of Fibroblasts of
Low and High Population Doubling Levels Grown in Collagen
Lattices", Mech. _Ageing & Dev. 17, 107-117 (1981). .
Bell, Eugene et al., "Development and Use of a Living Skin
Equivalent", J. Plastic & Reconstructive Surgery 67, 386-392
(1981). .
Elsdale et al., J. Cell. Bio., vol. 54 (1972), pp. 626-637. .
Ehrmann et al., J. Nat'l. Canc. Inst., vol. 16 (1956), pp.
1375-1403. .
Michalopoulos et al., Experm. Cell. Res., vol. 94 (1975), pp.
70-78..
|
Primary Examiner: Apley; Richard J.
Assistant Examiner: Isabelle; David J.
Attorney, Agent or Firm: Smith, Jr.; Arthur A. Brook; David
E. Reynolds; Leo R.
Parent Case Text
RELATED APPLICATION
This application is a continuation-in-part of Ser. No. 261,928
filed May 8, 1981, now U.S. Pat. No. 4,539,716 and is related to
Ser. No. 972,832 filed Dec. 26, 1978 and Ser. No. 245,536 filed
Mar. 19, 1981, both of which are now abandoned in favor of
continuation-in-part applications.
Claims
I claim:
1. In a method of producing, in vitro, a living multi-layered
tubular structure, comprising:
a. producing a first tubular layer by:
(1) forming a first aqueous mixture of collagen fibrils, nutrient
medium and a first cellular contractile agent capable of
interacting with collagen fibrils;
(2) introducing said first mixture into an annular casting
chamber;
(3) maintaining the annular casting chamber containing said first
mixture under conditions sufficient to allow a gel to form therein
and to allow radial contraction of the gel with expression of
aqueous liquid therefrom resulting from interaction of the cellular
contractile agent with collagen fibrils to thereby form a
contracted hydrated collagen lattice suitable as one layer of a
multi-layered tubular structure; and
(4) removing aqueous liquid expressed in the formation of said
first tubular layer from the annular casting chamber;
b. producing a second tubular layer outwardly of said first tubular
layer by:
(1) forming a second aqueous mixture of collagen fibrils, nutrient
medium and a second cellular contractile agent capable of
interacting with collagen fibrils;
(2) introducing said second mixture into the annular casting
chamber;
(3) maintaining the annular casting chamber containing said second
mixture under conditions sufficient to allow a gel to form therein
and to allow radial contraction of the gel with expression of
aqueous liquid therefrom resulting from interaction of the cellular
contractile agent with collagen fibrils to thereby form a
contracted hydrated collagen lattice suitable as another layer of a
multi-layered tubular structure; and
c. removing said multi-layered tubular structure from the annular
casting chamber;
The improvement comprising adding a reinforcing sleeve of inert
material to said living multi-layered tubular structure.
2. The improvement of claim 1 wherein said reinforcing sleeve of
inert material comprises a plastic mesh sleeve.
3. The improvement of claim 2 wherein said plastic mesh sleeve is
located between the first and second tubular layers of the living
multi-layered tubular structure.
4. The improvement of claim 3 wherein the plastic mesh sleeve is
attached by first slipping it on a tube and subsequently sliding
the tube over the first tubular structure and removing the tube
leaving the sleeve positioned over the first tubular structure.
5. The improvement of claim 3 wherein said plastic mesh sleeve is
pretreated to render it more electronegative.
6. The improvement of claim 5 wherein said plastic mesh screen is
pretreated by subjecting it to plasma.
7. A living multi-layered tubular structure produced by the
improvement of claim 1.
8. A living multi-layered tubular structure produced by the
improvement of claim 3.
9. In a method of producing a living prosthesis, in vitro,
comprising the steps of:
a. fabricating a cylindrical smooth muscle cell layer as
follows:
(aa) separately preparing (i) an aqueous acidic mixture comprising
nutrient medium and collagen fibrils and (ii) a mixture of smooth
muscle cells suspended in nutrient medium;
(ab) raising the pH of mixture (i) and quickly combining mixture
(i) and mixture (ii) and pouring the combined mixture into a
casting chamber having an inner core member and an outer
cylindrical wall structure to form a lattice;
(ac) incubating the lattice for a period sufficient to enable
collagen fibrils to be compacted by the cells so that aqueous
liquid is expressed out of the lattice as the lattice contracts
radially about the core;
(ad) removing aqueous liquid expressed in step (ac);
(ae) repeating steps (aa)-(ad) if additional layers of smooth
muscle cells are desired;
b. fabricating a layer containing fibroblast cells on said
cylindrical smooth muscle layer as follows:
(ba) separately preparing (i) an aqueous acidic mixture comprising
nutrient medium and collagen fibrils and (ii) a mixture of
fibroblast cells suspended in nutrient medium;
(bb) raising the pH of mixture (i) and quickly combining mixture
(i) and mixture (ii) and pouring the combined mixture into a
casting chamber having as an inner core member of cylindrical
smooth muscle cell layer and an outer cylindrical wall structure to
form a lattice;
(bc) incubating the lattice formed in step (bb) in accordance with
step (ac);
(bd) removing aqueous liquid expressed in step (bc);
c. lining the inner wall of the cylindrical smooth muscle cell
layer with living cells;
The improvement of including a plastic mesh within said living
prosthesis to thereby reinforce said living prosthesis and to
provide it with an increased degree of elasticity.
10. The improvement of claim 8 wherein said plastic mesh comprises
a sleeve positioned between the layers of smooth muscle cells and
fibroblast cells.
11. The improvement of claim 10 wherein said plastic mesh screen
comprises polyethylene terephthalate.
12. The improvement of claim 11 wherein said plastic mesh screen is
pretreated to make it more electronegative.
13. The improvement of claim 12 wherein said pretreatment is done
by exposing the plastic mesh screen to plasma.
14. A living multi-layered tubular structure produced by the
improvment of claim 9.
15. In a tubular prosthesis formed of multiple layers of hydrated
collagen lattices contracted with living cells:
The improvement comprising including a plastic mesh sleeve embedded
in the layers of said prosthesis to provide reinforcement and
elasticity thereto.
16. The improvement of claim 15 wherein said plastic mesh sleeve is
formed from polyethylene teraphthalate.
Description
TECHNICAL FIELD
This invention is in the field of biology and particularly relates
to the fabrication of living tissue in tubular form for various
applications such as capillaries, larger blood vessels and
glandular prosthesis.
BACKGROUND ART
Some of the material in the first of the referenced related
applications above has been published in the Proc. Natl. Acad. Sci.
USA Vol. 76 No. 3 pp 1274-1278 March 79 in an article entitled
"Production of a Tissue-Like Structure by Contraction of Collagen
Lattices by Human Fibroblasts of Different Proliferative Potential
In Vitro" by Bell et al. This article and the related applications
are mainly concerned by the fabrication of planar surfaces of
skin-like living tissue. This living tissue is produced in vitro by
forming a hydrated collagen lattice, containing a contractile
agent, such as fibroblast cells or blood platelets which contract
the lattice. This skin-like tissue is formed in a round or
rectangular vessel with, or without, a frame of stainless steel
mesh lying on the floor of the vessel. In its absence, the lattice
contracts in all dimensions; in its presence as the lattice sets it
becomes anchored to the mesh and contracts in the thickness
dimension only. The mesh, resembling a picture frame, holds the
lattice of living tissue within it. The contracted lattice, with or
without the stainless steel mesh frame, can be seeded with
epidermal cells from the potential graft recipient. When a sheet of
epidermal cells forms, the two layered skin equivalent is
grafted.
The resultant graft is unique as compared to any other graft
obtained from artificial skin since its basic organization is like
that of skin and its living constituent cells are donated by
potential graft recipients.
DISCLOSURE OF THE INVENTION
This invention relates to the casting of living collagen lattices
contracted by living cells, such as fibroblasts, smooth muscle
cells, or elements of cells such as blood platelets. In particular,
the lattices are cast into shapes which provide internal surface
areas and tubular shaped terminals, or end structures, particularly
effective for making connections, in vivo, with existing tubular
structures, such as capillaries, blood vessels and glandular
tissues.
The internal surface of the cast structure is lined with
specialized cells, depending on the function of the structure. For
example, endothelial cells are used for the internal surface of an
artery, vein, or other structures with internal surfaces.
Alternatively, in some applications it may be desirable to line the
internal surface with specialized cells having a predetermined
therapeutic value. For example, the inner surfaces of a capillary
bed may be lined with pancreatic .beta. cells to boost the insulin
supply in the blood. Pancreatic islets (islets of Langerhans),
hepatocytes or other types of glandular cells may also be used for
lining the inner surface of the vessel-equivalent structures.
In one embodiment, the structure is in the form of a tube, or
cylinder. The central core for forming the tube consists of
polyethelene or glass tubing. This core is axially centered within
a cylindrical mold. Suitable tissue forming constituents are poured
into the cylindrical mold. After a suitable period of time, the
tissue forming constituents contract the lattice and close in
around the central core. This procedure can be repeated as many
times as desired with the same or different cell types in the same
or different proportions to yield a multilayer tube. After each
layer contracts the fluid expressed from the contracting lattice is
poured off to accomodate the tissue forming constituents of the
next layer. The central core may then be removed and suitable
cells, predicated on the function of the cast structure, may then
be cultured on the inner surface of the hollow tissue cylinders, to
form, for example, a vessel-equivalent structure.
The fortuitous fact that the lattice contracts radially about the
central core structure to form tubes enables one to form various
shaped structures defined by the inner core surface. If, instead,
the lattice contracted in all directions, the resultant structure
would end up as a shapeless mass at the bottom of the mold. It is
also important to note that in the formation of vessel-equivalent
structure, in accordance with the invention, the sequential
addition of cells in an ordered pattern of layers is essential.
The vessel-equivalent structure thus far described is devoid of
elastin, the fibrous mucoprotein which is the major connective
tissue protein of elastic structures (e.g. large blood vessels).
Without this elastic property it is possible that the vessel could
burst under pressure. Since elastin is an extremely insoluble
substance it is difficult to directly incorporate elastin into the
molded tissue forming constituents previously described.
Accordingly, a plastic mesh may be optionally provided between two
layers or within a layer of the tissue forming constituents during
the molding process, as will be described in detail.
This mesh serves to reinforce the resultant vessel and at the same
time provide a degree of elasticity to the structure so that it may
expand and contract in the manner of a natural blood vessel having
elastin.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a perspective view of a first embodiment of the invention
showing the structure of the casting chamber.
FIG. 1A is a cross-sectional view of FIG. 1 showing a vessel as
cast.
FIG. 1B is a perspective view showing a plastic mesh on a support
tube which is used to position the mesh during casting.
FIG. 2 is a schematicized view showing the culturing apparatus of
the invention.
BEST MODE OF CARRYING OUT THE INVENTION
The following description generally relates to the casting of
cylindrical structures intended as prosthesis for vessels or
capillaries since such structures are commonly found in the human
body. However, other shapes may be conviently cast in accordance
with the teachings herein and the invention is not intended to be
limited to any particular shape or body structure.
FIG. 1 shows a preferred form of casting chamber for fabricating a
blood vessel-equivalent of living matter. The casting chamber 10
comprises a central rod or mandrel 12 disposed in a cylinder 16.
The central rod and cylinder are mounted on a base or stand 14. The
rod 12 is provided with three arms or spokes 18 at the top of the
rod for centering the rod within the cylinder 16.
The base is provided with an appropriate collar 20 to accept the
central rod 12. The outer cylinder has an internal diameter such
that when the arms 18 are disposed as shown and the central rod is
located in the collar 20, the rod 12 will be centered within
cylinder 16. The outer diameter of the rod 12 determines the inner
diameter of the cast vessel and for many applications would be in
the range of from 2-10 mm.
With the diameter of the central rod kept constant, the inner
diameter of cylinder 16 will determine the final thickness of the
cast layer, and typically may range from 1-4 cm to produce a final
thickness of about 0.5-2 mm, the final thickness being proportional
to the diameter. The height of the chamber determines the length of
the vessel and would typically be between 10-30 cm in height.
The casting chamber parts should be made from material which may be
readily cleaned and is autoclavable. Preferably, the cylinder 16
should be made from material which is clear and which will permit
diffusion of carbon dioxide and other gases. Thus, the rod 12 may
be made of glass or metal and the cylinder 16 should preferably be
made of autoclavable plastic, such as polycarbonate. The stand 14
may be made of glass, plastic or metal, such as stainless
steel.
The size and structure of blood vessels varies in accordance with
the function of the particular blood vessel. Blood vessels may be
generally characterized by their cellular composition and the
composition of the matrix or collagen lattice with which other
extracellular elements, such as elastin fibers and proteoglycans
are associated. The collagen, elastin, and proteoglycans are the
biosynthetic products of the cells in each of the layers.
The cell types are endothelial, smooth muscle, and fibroblasts
(called pericytes) and are found respectively in successive layers
from the lumen outward. In order to construct a particular type of
blood vessel, the respective layers may be laid down in order.
Alternatively, several can be laid down concurrently. All vessels
contain an inner endothelial lining. In an artery, for example,
smooth muscle surrounds the endothelium and the final outside layer
is made up of fibroblasts.
The process for fabricating the above described multilayered blood
vessel-equivalent will now be described in detail in connection
with FIGS. 1 and 2.
First, the smooth muscle layer is fabricated. A mixture of nutrient
medium (e.g. McCoy's medium containing fetal bovine serum) is
prepared in a flask. The ingredients are mixed in the following
ratio: 9.2 ml of 1.76.times.concentrate of McCoy's medium and 1.8
ml of fetal bovine serum. The pH is raised by addition of 1.0 ml of
0.1N NaOH. The foregoing mixture of medium and serum is poured onto
a dish in which 1.5 ml of native collagen in a 1-1000 acidic acid
solution has been prepared. About 250,000 cultured aorta smooth
muscle cells suspended in a 0.5 ml of McCoy's medium supplemented
with a 10% fetal bovine serum is quickly added. The above
constituents are mixed by swirling the dish and quickly pouring the
mixture into the casting chamber. The chamber is then placed in a
humidified 5% CO.sub.2, 95% air incubator at 37.degree. C. for 3
days.
A collagen lattice or gel forms immediately on casting the mixture.
The collagen fibrils are gradually compacted by the cells so that
fluid is squeezed out of the lattice. The result is contraction of
the collagen lattice around the central core or rod 12. After 3
days in the incubator, the smooth muscle layer will have set in a
cylindrical structure having sufficient structural integrity to
simulate, or replicate, the smooth muscle layer of a typical blood
vessel. If a second layer is to be applied, the fluid expressed
during contraction of the first lattice is poured off and a second
complete mixture of all ingredients is added to replace the fluid.
The process may be repeated as many times as desired to give a
multilayered structure. The layers may be poured simultaneously
with a removable separation or sleeve (not shown) between them. As
soon as gelation begins the sleeve is removed.
Optionally, after the smooth muscle layer cylinder has been cast,
it may be desirable to provide a plastic mesh sleeve 11 about the
outer surface of the smooth muscle layer cylinder or the mesh may
be embedded in the smooth muscle layer. This mesh will serve to
reinforce the resulting structure and provide some degree of
elasticity so that the resulting structure will be better able to
withstand the pressures it will be subjected to in use. Meadox
Medicals, Inc., 103 Bauer Drive, Oaklane, N.J. 07436, supplies a
Dacron.RTM. mesh sleeve, Part No. 01H183, which has proved
particularly suitable for this purpose. Other suitable meshes are
readily available in various inert plastics, such as Teflon.RTM.,
nylon, etc. and the invention is not to be limited to a particular
plastic material. Preferably, the mesh should be treated to render
it more electronegative by, for example, subjecting it to plasma.
This results in better cell attachment to the plastic sleeve and
hence an increase in the strength of the resultant structure.
The sleeve 11 should be placed on the smooth muscle cell cylinder
by first disposing the sleeve 11 on metal tube 15 (as shown in FIG.
1B) which has an inner diameter larger than the outer diameter of
the smooth muscle cell cylinder. The tube 15, with the sleeve on
the exterior, is then slipped over the smooth muscle cell cylinder,
a portion of the sleeve is then pulled off the tube 15 and onto the
smooth muscle cell cylinder and held there while the tube 15 is
slipped off the smooth muscle cell cylinder. This procedure
minimizes damage to the exterior surfaces of the smooth muscle cell
cylinder while attaching the sleeve.
Next, a fibroblast layer may be cast around the inner smooth muscle
layer(s) and sleeve 11 so as to completely enclose the sleeve 11,
as shown in FIG. 1A. In this process, the ingredients described
above in connection with the fabrication of a smooth muscle layer
are used to constitute a fibroblast layer, except that cultured
aorta fibroblasts are substituted for the smooth muscle cells. The
incubation period for the fibroblast layer may be 2 days to a
week.
The resultant multi-layered structure consisting of inner smooth
muscle layer(s) and an outer fibroblast layer with a mesh sleeve
sandwiched between the two layers is now ready to be cultured with
an inner endothelial lining of living endothelial cells. To perform
this step the cylindrical tissue tube of several layers is slipped
off the casting rod 12 to receive the endothelial cells as a
suspension. It is supported in the culturing apparatus shown in
FIG. 2.
The apparatus of FIG. 2 comprises a transparent chamber 24, within
which a rotatable rod 26 is inserted at one end and a rotatable
tube 36 is inserted at the opposite end. The tube 36 and rod 26 are
tied together by wire frame member 30 such that when the rod 26 is
rotated, the tube 36 will rotate in unison in the same direction.
Rod 26 is coupled to motor 28 such that when motor 28 is energized
the rod 26 will rotate in the direction shown by the arrow.
Preferably, the rod is attached to the motor in such a way that the
length of the rod inserted into the chamber 24 may be adjusted in
accordance with the length of the vessel-equivalent 44 being
supported within the culture chamber 24. This may be accomplished
by a rack and pinion device or other such variable length means
(not shown).
Rod 26 is provided at one end with a nipple 32 to which a vessel 44
(such as the structure previously described in connection with
FIGS. 1, 1A and 1B comprising an inner cylinder smooth muscle cell
layer, and an outer cylinder of fibroblast cells with a mesh sleeve
sandwiched between) may be attached. Similarly, tube 36 is provided
with a complementary nipple 34 to which the opposite end of the
vessel 44 may be attached. In this manner, the vessel 44 is
suspended between the rod 26 and tube 36 and a culture medium may
be introduced from reservoir 42 through tubing 40 and fixed
connecting tube 38, through tube 36 and into the interior lining of
blood vessel-equivalent 44. It should be understood that
water-tight seal bearings (not shown) are provided at both ends of
chamber 24 to permit the rod and tube to be inserted into the
chamber.
Reservoir 42 is supplied with a suspension of about 200,000
cultured aorta or other endothelial cells in McCoy's medium
supplemented with a 20% fetal bovine serum. This mixture is fed by
hydrostatic pressure from the reservoir into the vessel 44 as
previously mentioned. Next, the vessel 44 is slowly rotated by
means of motor 28 which preferably runs at a speed of between 0.1
and 1 r.p.m. Rotation of the vessel 44 enables distribution of the
endothelial cells evenly on the inner lining of the vessel and the
hydrostatic pressure head from the reservoir enables the lumen, or
inner opening, of the vessel-equivalent to remain open. It should
be emphasized that the above procedures are intended to be carried
out asceptically.
EQUIVALENTS
Those skilled in the art will recognize or be able to ascertain,
using no more than routine experimentation, other equivalents for
the specific reactants, steps and techniques, etc. described
herein. Such equivalents are intended to be included within the
scope of the following claims.
* * * * *